Human cytomegalovirus (HCMV) is among the most important opportunistic pathogens encountered in patients with AIDS. For example, HCMV infection is among the most common cause of oral diseases associated with HIV-positive individuals. Blocking viral lytic replication systemically as well as locally in the infected tissues is central for the treatment of HCMV systemic infections as well as viral-associated oral diseases. The emergence of drug-resistant HCMV strains has posed a need to develop new drugs and novel strategies to combat HCMV infections. The goal of the proposed research is to develop small novel RNA molecules as anti-HCMV therapeutic agents by directing endogenous RNase P to hydrolyze a HCMV essential mRNA and inhibit viral gene expression and replication. These RNA molecules, termed as external guide sequences (EGS), consist of a sequence antisense to the target mRNA and a sequence that guides RNase P to cleave the target. Previous studies have shown that custom-designed EGSs can target both cellular and viral mRNAs for RNase P degradation and inhibit their expression in tissue culture. However, the mechanism of how EGSs interact with the mRNA substrates and RNase P to achieve efficient and specific cleavage has not been extensively studied. Currently it is unknown whether the EGS technology is also effective in modulating gene expression in animal models in vivo. Equally unclear is the specificity and antiviral activity of EGSs in clinically relevant human cells. To address these issues, we propose to generate highly active and sequence-specific EGSs that direct RNase P to cleave the mRNAs encoding viral essential capsid scaffolding protein and UL79. Initially, how the generated EGS RNAs efficiently and specifically induce RNase P for cleavage will be studied in vitro by biochemical approaches. Then, whether these EGSs effectively abolish HCMV replication in human fibroblasts and cells differentiated from CD34+ progenitor cells will be determined, and the effects of the expression of EGSs on the proliferation and differentiation of these cells will be investigated. Moreover, the selected EGSs will be used as a gene targeting tool to investigate the function roles of UL79 in HCMV replication. Finally, using murine CMV infection of mice as the model system, we will determine whether EGSs are effective in blocking viral infection and pathogenesis in vivo in animals. The proposed study will generate novel and exciting EGSs that can serve as lead compounds for anti-HCMV therapeutic development. Furthermore, our study will provide the first direct evidence of whether the EGS-based technology is effective in modulating gene expression in vivo in animals. These results will facilitate the development of these novel RNA-based agents for treatment and studies of AIDS-associated opportunistic infections, including those caused by HCMV.